The present invention relates to a method of introducing molecules into cells using a photosensitising agent and irradiation of cells with light of a wavelength effective to activate the photosensitising agent, wherein the molecule to be introduced is associated with a viral carrier and in particular an adenovirus carrier. The present invention further relates to the use of this method in gene therapy.
Gene therapy, i.e. the genetic modification of the cells of a patient in order to combat disease, is recognized as having a large therapeutic potential for treating a variety of diseases, such as cancer, infectious diseases including viral and bacterial infections, cardiovascular disease, inherited disorders such as cystic fibrosis, immune system disorders and many other conditions. The clinical development of gene therapy is, however, still faced with many unsolved challenges, of which one of the most important is to find methods for efficient and specific delivery of therapeutic genes to the target cells in vivo (Verma & Somia, 1997, Nature, vol. 389, 239-242 and Anderson, 1998, Nature vol. 392, 25-30).
Gene therapy can involve many different possible approaches and can involve transfer of cloned human genes or gene segments, double stranded human genes or gene segments, genes from other genomes and organisms, oligonucleotides and various artificial genes or fragments thereof such as antisense genes.
In current methods many different carriers or vectors have been suggested for use in achieving gene transfer in gene therapy. As examples polycationic compounds, cationic lipids and viral systems can be mentioned, but as yet in vivo gene therapy has met with little success. Among the many known drawbacks of the current methods are low serum stability of the vector, limited specificity in gene delivery, low efficiency in gene delivery etc. The use of viral carriers has been approached with particular caution due to the introduction of viral elements into hosts which can cause adverse effects such as inflammation, which is not offset by enhanced transfer compared to other methods.
The majority of molecules do not readily penetrate cell membranes. Methods for introducing molecules into the cytosol of living cells are known in the art and are useful tools for manipulating and studying biological processes. Among the most commonly used methods are microinjection, red blood cell ghost-mediated fusion and liposome fusion, osmotic lysis of pinosomes, scrape loading, electroporation, calcium phosphate and virus-mediated transfection. These techniques are useful for manipulating cells in culture, although in many cases they may be impractical, time consuming, inefficient or they may induce significant cell death. Thus such techniques are not optimal for use in biological or medical research, or in therapies, where they are often not sufficiently efficient, may have intolerable toxic effects or may not be applicable for technical reasons.
It is well known that porphyrins and many other photosensitizing compounds may induce cytotoxic effects on cells and tissues. These effects are based upon the fact that upon exposure to light the photosensitizing compound may become toxic or may release toxic substances such as singlet oxygen or other oxidising species which are damaging to cellular material or biomolecules, including the membranes of cells and cell structures, and such cellular or membrane damage may eventually kill the cells. These effects have been utilised in the treatment of various abnormalities or disorders, including especially neoplastic diseases. The treatment is named photodynamic therapy (PDT) and involves the administration of photosensitizing (photochemotherapeutic) agents to the affected area of the body, followed by exposure to photoactivating light in order to activate the photosensitizing agents and convert them into cytotoxic form, whereby the affected cells are killed or their proliferative potential diminished. Photosensitizing agents are known which will localise preferentially or selectively to the desired target site e.g. to a tumour or other lesion.
A range of photosensitizing agents are known, including notably the psoralens, the porphyrins, the chlorins and the phthalocyanins. Such drugs become toxic when exposed to light.
Porphyrin photosensitisers act indirectly by generation of toxic oxygen species, and are regarded as particularly favourable candidates for PDT. Porphyrins are naturally occurring precursors in the synthesis of heme. In particular, heme is produced when iron (Fe3+) is incorporated in protoporphyrin IX (PpIX) by the action of the enzyme ferrochelatase. PpIX is an extremely potent photosensitizer, whereas heme has no photosensitizing effect. A variety of porphyrin-based or porphyrin-related photosensitisers are known in the art and described in the literature.
The cytotoxic effect is mediated mainly through the formation of singlet oxygen. This reactive intermediate has a very short lifetime in cells (<0.04 μs). Thus, the primary cytotoxic effect of PDT is executed during light exposure and very close to the sites of formation of 1O2. 1O2 reacts with and oxidizes proteins (histidine, tryptophan, methionine, cysteine, tyrosine), DNA (guanine), unsaturated fatty acids and cholesterol. One of the advantages of PDT is that tissues unexposed to light may be left unaffected ie. that a selective PDT effect may be obtained. There is extensive documentation regarding use of PDT to destroy unwanted cell populations, for example neoplastic cells. The patent literature describes a number of photodynamic compounds, alone or conjugated with targeting agents, e.g. immunoglobulins directed to neoplastic cell receptor determinants, making the complex more cell specific. Certain photochemical compounds, such as hematoporphyrin derivatives, have furthermore an inherent ability to localise in malignant cells. Such methods and compounds, are described in the Norwegian patent No. 173319 and in Norwegian patent applications Nos. 90 0731, 176 645, 176 947, 180 742, 176 786, 301 981, 30 0499 and 89 1491. Such PDT methods are thus dependent on the destruction of cell structures leading to cell death.
WO 96/07432 or the copending application WO 00/54802 on the other hand, are concerned with methods which use the photodynamic effect as a mechanism for introducing otherwise membrane-impermeable molecules into the cytosol of a cell in a manner which does not necessarily result in widespread cell destruction or cell death. In these methods, the molecule to be internalised and a photosensitising compound are applied simultaneously or in sequence to the cells, upon which the photosensitizing compound and the molecule are endocytosed or in other ways translocated into endosomes, lysosomes or other intracellular membrane restricted compartments.
The molecule to be translocated and the photosensitising compound are applied to the cells together or sequentially (preferably separately and sequentially) and are taken up by the cell together into the same intracellular compartments (i.e. are co-translocated). The molecule to be internalised within the cell is then released by exposure of the cells to light of suitable wavelengths to activate the photosensitising compound which in turn leads to the disruption of the intracellular compartment membranes and the subsequent release of the molecule, which is located in the same compartment as the photosensitizing agent, into the cytosol. This method was termed “photochemical internalisation” or PCI. Thus, in these methods the final step of exposing the cells to light results in the molecule in question being released from the same intracellular compartment as the photosensitizing agent and becoming present in the cytosol.
It was believed that in order for this technique to be effective it was essential that both the photosensitising compound and the molecule to be released into the cytosol were present in the same intracellular compartments when irradiation was performed. However, it has since been found that molecules can be introduced into the cytosol of cells by similar PCI methods but where the exposure of the cells to light is not necessarily the final step and the methods are not dependent on the transfer molecule and the photosensitizing agent being located in the same intracellular compartments at the time of light exposure. In such methods the photosensitising agent may be contacted with the cells and activated by irradiation before the molecule to be internalised and thus delivered to the cytosol is brought into contact with the cells. Thus, despite the fact that the molecule to be internalised and the photosensitising agent are not necessarily localised in the same intracellular compartments at the time of light exposure, the molecule still enters the cell and is delivered to the cytosol. These results are described in detail in the co-pending international application (filed on 29 Nov. 2001 in the name of The Norwegian Radium Hospital Research Foundation, entitled “Method”), a copy of which is appended hereto and is incorporated herein by reference.
Surprisingly it has now been found that the use of PCI techniques in combination with viral vectors can substantially improve the virus mediated gene delivery to a cell. Since photochemical treatments are in clinical use (Dougherty et al, 1998, J. Natl. Cancer Inst, vol. 90, 889-905), and generally are very specific and have few side effects, the technology described has a clear potential for improving both the efficiency and the specificity of in vivo gene therapy.